The objectives of this proposal are to perform molecular and mechanistic analyses of the Vitamin K-dependent carboxylase, a liver endoplasmic reticular enzyme that carries out post-translational carboxylation of the most amino proximal 10-12 glutamyl residues in proteins such as prothrombin and Factor IX involved in blood coagulation. The resultant gamma-carboxyglutamyl (Gla) residues are high affinity calcium binding sites crucial for platelet membrane binding and for initiation of coagulation cascades. Now that this unusual O2 and dihydro vitamin K- utilizing enzyme has been purified, cloned, sequenced, and recombinant enzyme expressed (usefully for purification in baculovirus), we plan to perform analyses on molecular recognition of specific proteins and on catalytic mechanism. Specific aims include: recognition of substrates at both the upstream propeptide recognition site (gamma-carboxylation recognition site) and the active site, directionality and processivity of glutamyl residue carboxylation and quantitation of binding of enzyme to peptide substrates containing 1 to 10 glutamates (28mers to 59mers) by surface plasmon resonance and capillary zone electrophoresis techniques. Analysis of mechanism will include characterization of the enzyme as a hydroquinone epoxidase, and study of the timing of gamma-CH cleavage and carboxylation relative to KH2 and O2 reaction. Mechanistic insight may permit rational design of carboxylase inhibitors that complement such oral anticoagulants as the coumarins which inhibit the subsequent enzyme that recycles vitamin K epoxide.